The objectives of this proposal are to identify and determine the function of rapidly synthesized labile proteins induced by ACTH in the adrenal cortex, to determine the mechanism by which the synthesis or activity of such proteins are controlled and to study the effects of potential modifiers of cytochrome P450 on corticosteroidogenesis and adrenal cortical metabolism. Rapidly synthesized ACTH-dependent proteins will be identified by double-labelling of proteins in either adrenal slices, isolated cells or mouse adrenal tumor cells and isolation attempted from appropriate tissues by a combination of ion exchange, gel filtration chromatography and gel isoelectric focusing. The effects of such proteins as well as other potential modifiers of corticosteroidogenesis will be studied by kinetic analysis of steroid binding and hydroxylation by isolated purified cytochrome P450s from bovine adrenals and by adrenal cortical mitochondria and microsomes from cycloheximide treated or hypophysectomized rats. EPR and dual wave length visible spectroscopy will be used to study the kinetics of corticosteroid intermediate binding to cytochrome P450s and the steady levels of steroid-bound P450s in mitochondria and microsomes from adrenals of rats receiving various treatments in vivo. In addition, the relationship of the ACTH receptor-cAMP-protein kinase regulatory system to ACTH induced proteins will be studied in a mouse adrenal tumor cell culture and in normal and abnormal human adrenals.